EFAR - Escola de Farmácia
URI permanente desta comunidadehttp://www.hml.repositorio.ufop.br/handle/123456789/451
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O curso de Farmácia em Ouro Preto foi criado em 1839, sendo a mais antiga Escola de Farmácia da América Latina.
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Item A specific Leishmania infantum polyepitope vaccine triggers Th1-type immune response and protects against experimental visceral leishmaniasis.(2022) Ostolin, Thais Lopes Valentim Di Paschoale; Gusmão, Miriã Rodrigues; Mathias, Fernando Augusto Siqueira; Cardoso, Jamille Mirelle de Oliveira; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Ruiz, Jeronimo Conceição; Resende, Daniela de Melo; Brito, Rory Cristiane Fortes de; Reis, Alexandre BarbosaThe development of an immunogenic, effective, and safe vaccine is essential as an alternative for disease control. The present study aimed to evaluate the immunogenicity and efficacy potential of a polyepitope T-cell antigen candidate against visceral leishmaniasis in a murine model. BALB/c mice were immunized with three doses subcutaneously with Poly-T Leish alone or adjuvanted with Saponin plus Monophosphoryl lipid A, with 15-day intervals between doses, and challenged with 107 stationary-phase Leishmania infantum promastigotes via tail vein. Immunogenicity and parasitism in spleen and liver of immunized mice were evaluated 45 days postchallenge. Our results revealed that the immunization with Poly-T Leish and Poly-T Leish/SM increases the percentage of specific T (CD4+ and CD8+) lymphocytes proliferation in vitro after antigen-specific stimulation. Also, Poly-T Leish and Poly-T Leish/SM groups showed a high percentage of IFN-γ and TNF-α-producing T cells, meanwhile, the Poly-T Leish/SM group also showed an increased percentage of multifunctional T cells producing double and triple-positive (IFN-γ+TNF-α+IL-2+) cytokines. The immunization with Poly-T Leish or Poly-T Leish/ SM stimulated a decreased IL-4 and IL-10 compared to the Saline and adjuvant group. Poly-T Leish/SM immunized mice exhibit a noteworthy reduction in the parasite burden (spleen and liver) through real-time PCR (96%). Moreover, we observed higher nitrite secretion in 120-hour stimulated-culture supernatant using Griess method. We demonstrated that the Poly-T Leish/SM candidate was potentially immunogenic, providing enhancement of protective immune mechanisms, and conferred protection reducing parasitism. Our candidate was considered potential against visceral leishmaniasis, and eventually, could be tested in phase I and II clinical trials in dogs.Item In vitro infectivity of strains isolated from dogs naturally infected with Leishmania infantum present a distinct pathogenic profile in hamsters.(2020) Resende, Lucilene Aparecida; Soares, Rodrigo Dian de Oliveira Aguiar; Moreira, Nádia das Dores; Ferreira, Sidney de Almeida; Lanna, Mariana Ferreira; Cardoso, Jamille Mirelle de Oliveira; Mathias, Fernando Augusto Siqueira; Vital, Wendel Coura; Mariano, Reysla Maria da Silveira; Leite, Jaqueline Costa; Silveira, Patricia; Carvalho, Tatiane Furtado de; Santos, Renato Lima; Lemos, Denise da Silveira; Martins Filho, Olindo Assis; Dutra, Walderez Ornelas; Reis, Alexandre Barbosa; Giunchetti, Rodolfo CordeiroVisceral leishmaniasis (VL) is a severe disease caused by Leishmania infantum. Dogs are the parasite’s main reservoir, favoring its transmission in the urban environment. The analysis of L. infantum from infected dogs contributes to the identification of more virulent parasites, thereby supporting basic and applied studies such as vaccinal and therapeutic strategies. We proposed the in vitro and in vivo characterization of L. infantum strains from naturally infected dogs from a VL endemic area based on an infectivity and pathogenicity analysis. DH82 canine macrophages were infected in vitro with different strains for infectivity analysis, showing distinct infectivity profiles. The strains that showed greater and lesser infectivity using in vitro analyses (616 and 614, respectively) were used to infect hamsters for pathogenicity analysis. The group infected with strain 616 showed 100% survival while the group infected with strain 614 showed 50% after seven months of follow up. Furthermore, the 614 strain induced more noticeable clinicopathological changes and biochemical abnormalities in liver function, along with high inflammation and parasite load in the liver and spleen. We confirmed high variability of infectivity and pathogenicity in L. infantum strains from infected dogs. The results support the belief that screening for L. infantum infectivity using in vitro experiments is inadequate when it comes to selecting the most pathogenic strain.Item Mixed formulation of conventional and pegylated meglumine antimoniate-containing liposomes reduces inflammatory process and parasite burden in Leishmania infantum-infected BALB/c mice.(2017) Reis, Levi Eduardo Soares; Brito, Rory Cristiane Fortes de; Cardoso, Jamille Mirelle de Oliveira; Mathias, Fernando Augusto Siqueira; Soares, Rodrigo Dian de Oliveira Aguiar; Carneiro, Cláudia Martins; Vieira, Paula Melo de Abreu; Ramos, Guilherme Santos; Frezard, Frederic Jean Georges; Roatt, Bruno Mendes; Reis, Alexandre BarbosaPentavalent antimonial has been the first choice treatment for visceral leishmaniasis; however, it has several side effects that leads to low adherence to treatment. Liposome-encapsulated meglumine antimoniate (MA) arises as an important strategy for chemotherapy enhancement. We evaluated the immunopathological changes using the mixture of conventional and pegylated liposomes with MA. The mice were infected with Leishmania infantum and a single-dose treatment regimen. Comparison was made with groups treated with saline, empty liposomes, free MA, and a liposomal formulation of MA (Lipo MA). Histopathological analyses demonstrated that animals treated with Lipo MA showed a significant decrease in the inflammatory process and the absence of granulomas. The in vitro stimulation of splenocytes showed a significant increase of gamma interferon (IFN-γ) produced by CD8+ T cells and a decrease in interleukin-10 (IL-10) produced by CD4+ and CD8+ T cells in the Lipo MA. Furthermore, the Lipo MA group showed an increase in the IFN-γ/IL-10 ratio in both CD4+ and CD8+ T cell subsets. According to the parasite load evaluation using quantitative PCR, the Lipo MA group showed no L. infantum DNA in the spleen (0.0%) and 41.4% in the liver. In addition, we detected a low positive correlation between parasitism and histopathology findings (inflammatory process and granuloma formation). Thus, our results confirmed that Lipo MA is a promising antileishmanial formulation able to reduce the inflammatory response and induce a type 1 immune response, accompanied by a significant reduction of the parasite burden into hepatic and splenic compartments in treated animals.Item Peptide vaccines for leishmaniasis.(2018) Brito, Rory Cristiane Fortes de; Cardoso, Jamille Mirelle de Oliveira; Reis, Levi Eduardo Soares; Vieira, João Filipe Pereira; Mathias, Fernando Augusto Siqueira; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Ruiz, Jeronimo Conceição; Resende, Daniela de Melo; Reis, Alexandre BarbosaDue to an increase in the incidence of leishmaniases worldwide, the development of new strategies such as prophylactic vaccines to prevent infection and decrease the disease have become a high priority. Classic vaccines against leishmaniases were based on live or attenuated parasites or their subunits. Nevertheless, the use of whole parasite or their subunits for vaccine production has numerous disadvantages. Therefore, the use of Leishmania peptides to design more specific vaccines against leishmaniases seems promising. Moreover, peptides have several benefits in comparison with other kinds of antigens, for instance, good stability, absence of potentially damaging materials, antigen low complexity, and low-cost to scale up. By contrast, peptides are poor immunogenic alone, and they need to be delivered correctly. In this context, several approaches described in this review are useful to solve these drawbacks. Approaches, such as, peptides in combination with potent adjuvants, cellular vaccinations, adenovirus, polyepitopes, or DNA vaccines have been used to develop peptide-based vaccines. Recent advancements in peptide vaccine design, chimeric, or polypeptide vaccines and nanovaccines based on particles attached or formulated with antigenic components or peptides have been increasingly employed to drive a specific immune response. In this review, we briefly summarize the old, current, and future stands on peptide-based vaccines, describing the disadvantages and benefits associated with them. We also propose possible approaches to overcome the related weaknesses of synthetic vaccines and suggest future guidelines for their development.Item Canine visceral leishmaniasis follow-up : a new anti-IgG serological test more sensitive than ITS-1 conventional PCR.(2017) Faria, Angélica Rosa; Pires, Simone da Fonseca; Reis, Alexandre Barbosa; Vital, Wendel Coura; Silveira, Júlia Angélica Gonçalves da; Sousa, Gabriela Matos de; Bueno, Melissa Luíza Couto; Gazzinelli, Ricardo Tostes; Andrade, Hélida Monteiro deVisceral leishmaniasis (VL) is a neglected tropical disease with dogs serving as reservoirs for one of its etiological agents, Leishmania infantum. In Brazil, VL control involves culling of seropositive dogs, among other actions. However, the most employed serological tests lack accuracy, and are not able to detect canine visceral leishmaniasis (CVL) during the early stages of infection. Early detection of CVL is highly desirable in order to shorten the contact time between the infected reservoirs and the vectors. In this study, we investigated the ability of two multiepitope proteins, PQ10 and PQ20, to detect CVL at earlier stages than currently employed methods, including ITS-1 conventional PCR. Using serum samples from naturally infected dogs, we observed that ELISAPQ10 and ELISA-PQ20 were able to detect Leishmania infection at earlier time points as compared with kDNA PCR-RFLP in anti-IgG and anti-IgM assays. Using sera from experimentally infected dogs, we monitored seroconversion using multiepitope proteins, ELISA-crude antigen, as well as ITS-1 conventional and real-time PCR. While seroconversion was detected by ELISA-crude antigen in 16.6% of the dogs, multiepitope proteins were able to detect seroconversion in more than 80% of them. Moreover, the ability of ELISA-PQ10 and ELISA-PQ20 to detect Leishmania infection at earlier time points as compared with conventional PCR was also confirmed in experimental infection dogs’ sera. Immunofluorescence to Babesia canis and Ehrlichia canis did not show crossreactions with ELISA-PQ10/PQ20 positive samples. Results of real-time PCR and ELISA with multiepitope proteins were very similar, with concordances between 80 and 100%. Furthermore, our findings indicated that PQ10 and PQ20 immunoassays can be related to parasite load. ELISA-PQ10 and ELISA-PQ20 are more sensitive diagnostic tools for early CVL detection as compared with other methods They could potentially be used in screening tests due to easy execution and low costs facilities.Item An antigenic domain within a catalytically active leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies.(2013) Maia, Ana Carolina Ribeiro Gomes; Porcino, Gabriane Nascimento; Detoni, Michelle de Lima; Emídio, Nayara Braga; Marconato, Danielle Gomes; Pinto, Priscila de Faria; Fessel, Melissa Regina; Reis, Alexandre Barbosa; Juliano Neto, Luiz; Juliano, Maria Aparecida; Marques, Marcos José; Vasconcelos, Eveline GomesWeidentified a shared B domainwithin nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50 kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived fromthe potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived fromthe Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83–122). By immunoprecipitation assays andWestern blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87–99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.Item Autochthonous canine visceral leishmaniasis in a non-endemic area : Bom Sucesso, Minas Gerais State, Brazil.(2008) Silva, Marcio Roberto; Marques, Marcos José; Romanha, Alvaro José; Santa Rosa, Idael Christiano de Almeida; Carneiro, Cláudia Martins; Reis, Alexandre BarbosaO presente trabalho descreve inicialmente um cão com sintomas característicos de leishmaniose visceral. Amostra de soro desse cão foi positiva por imunofluorescência indireta (IFI) conduzida no IgG total anti- Leishmania em 1999. Além disso, tecidos desse cão foram positivos por reação em cadeia pela polimerase (PCR) conduzida em 2004, identificando DNA de Leishmania no cerebelo, fígado, rim e intestino. Esta é a primeira vez que um cão com leishmaniose visceral autóctone foi descrito no Município de Bom Sucesso, Minas Gerais, Brasil. O achado desse cão reagente à IFI levou a uma investigação epidemiológica nesse município. Essa investigação foi conduzida de março de 1999 a dezembro de 2005. Vinte e dois de um total de 734 (3%) cães examinados foram reagentes à IFI. Entre os 22 cães IFI reagentes, seis apresentaram sintomas característicos de leishmaniose visceral canina. Os resultados desta investigação epidemiológica foram enviados às autoridades locais e estaduais de saúde pública requerendo medidas preventivas e de controle para leishmaniose visceral de forma a interromper a transmissão da doença e evitar a ocorrência de casos humanos.Item Development of a label-free immunosensor based on surface plasmon resonance technique for the detection of anti-Leishmania infantum antibodies in canine serum.(2013) Souto, Dênio Emanuel Pires; Silva, Jussara Vieira; Martins, Helen Rodrigues; Reis, Alexandre Barbosa; Luz, Rita de Cássia Silva; Kubota, Lauro Tatsuo; Damos, Flavio SantosIn this work ,a surface Plasmon resonance (SPR) immune sensor was developed using an 11-mercaptoundecanoic acid (11-MUA) modified gold SPR sensor chip for the detection of anti- Leishmania infantum antibodies. The soluble antigens of L. infantum were securely immobilized on an SPR gold disk byan 11-MUA self-assembled monolayer. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) techniques were employed in the characterization of the antigenim mobilization. After the immune sensor construction, canine serum positive for visceral leishmaniasis was added to its surface and showed significant variation in the SPR angle, indicating excellent sensitivity of the technique for antigen–antibody interaction detection. Moreover, the addition of negative serum was accompanied by as maller response, demonstrating that the immunosensor shows good specificity against anti-L. infantum antibodies. Therefore, this work demonstrates the success ful development of an SPR sensor for anti- L. infantum antibodies detection in short time, showing a great perspective as asensing system of visceral leishmania sisinende micregions.Item Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations.(2007) Ferreira, Eduardo de Castro; Lana, Marta de; Carneiro, Mariângela; Reis, Alexandre Barbosa; Paes, Daniela Vieira; Silva, Eduardo Sérgio da; Schalling, Henk; Gontijo, Célia Maria FerreiraThree serological methods, indirect fluorescent immunoassay (IFI), enzyme-linked immunosorbent assay (ELISA) and direct agglutination test (DAT) that are commonly employed in the diagnosis of canine visceral leishmaniasis (CVL), have been assessed. A total of 234 domestic dogs, drawn from an area in the municipality of Belo Horizonte, Minas Gerais, Brazil, endemic for visceral leishmaniasis, were submitted to clinical and parasitological examinations and serological assay. Sera collected from confirmed non-infected dogs (n = 20), and from dogs with other parasitic diseases including Trypanosoma cruzi ( n = 7), Leishmania braziliensis ( n = 5), Toxoplasma gondii ( n = 5) and Ehrlichia canis ( n = 3), were also included in the study. IFI presented a lower sensitivity (72%) than ELISA (95%), although the specificities of these assays were low (52 and 64%, respectively) and both exhibited cross-reactivity with sera from dogs infected with T. cruzi , L. braziliensis and E. canis. In contrast, DAT exhibited a high sensitivity (93%) and a high specificity (95%) and cross-reacted with only one serum sample derived from anE. canis-infected dog. The reproducibilities of the ELISA and DAT assays were excellent, whilst that of IFI was considered to be acceptable. The results produced by ELISA and DAT were in complete agreement, those between ELISA and IFI were at an acceptable level of agreement, whilst the concurrence between the IFI and DAT results were either acceptable or poor depending on the clinical conditions of the group of dogs examined. Since there is no readily accessible method for the diagnosis of CVL that offers 100% specificity and sensitivity, the choice of technique employed must depend on the aim of the investigation.Item Analysis of the cytokine profile in spleen cells from dogs naturally infected by Leishmania chagasi.(2007) Lage, Ramon Silva; Oliveira, Guilherme Corrêa de; Busek, Solange Cristina Uber; Guerra, Luanda Liboreiro; Giunchetti, Rodolfo Cordeiro; Oliveira, Rodrigo Corrêa de; Reis, Alexandre BarbosaRecent studies suggest that asymptomatic dogs infected with canine visceral leishmaniasis (CVL) develop a Th1 immunological profile whilst oligosymptomatic and symptomatic CVL-infected animals present a Th2 profile. In the present study, an RT-PCR method has been standardised and employed to evaluate the frequency and the semi-quantitative level of expression of the cytokines IL-4, IL-10, IL-12, INF- g and TNF- a in splenocytes of 30 dogs naturally infected with Leishmania chagasiand of 7 non-infected dogs (NID). An increase in the level of expression of IL-12 ( p = 0.059) was detected in all CVL-infected dogs compared with NID. In dogs exhibiting high parasitism, the frequency of expression of IL-10 was higher ( p = 0.011) than in animals presenting low parasitism or medium parasitism (MP) and in NID animals, whilst the level of expression of IL-10 was higher (p = 0.0094) than in animals exhibiting MP and in the NID group. Positive correlations between the levels of expression of IL-10 with respect to the progression of the disease (IL-10: r = 0.3510; p = 0.0337) and the levels of expression of IL-10 and INF- g increase in parasitism (IL-10: r = 0.3428; p = 0.0438 and INF- g: r = 0.4690; p = 0.0045) were observed. Such data suggest that CVL is marked by a balanced production of Th1 and Th2 cytokines, with a predominant accumulation of IL-10 as a consequence of an increase in parasitic load and progression of the disease, and INF- g was related with the increase in parasitic load.